Microphotometrical image analysis of the subtelomeric region of T-banded endoreduplicated chromosomes of Chinese hamster ovary cells

Microphotometrical image analysis of the subtelomeric region of T-banded endoreduplicated chromosomes of Chinese hamster ovary cells^*

Máximo E.Drets and Marcos Mendizábal

Division of Human Cytogenetics and Quantitative Microscopy, Instituto de Investigaciones Biológicas Clemente Estable, Avda. Italia 3318, 11600 Montevideo, Uruguay.

ABSTRACT

Microphotometrical scanning and computer graphic image analysis were carried out to detect the distribution of chromatin densities in subtelomeric segments of T-banded endoreduplicated chromosomes of Chinese hamster ovary (CHO) cells. Chromatin density patterns detected with this method were similar to those previously found in CHO and normal human chromosomes. The highest chromatin densities were considered as marker segments which led to the detection of reciprocal changes of position in endoreduplicated chromosomes during cell spreading on the slide. The problem of the subtelomeric T-banding density patterns found in the endoreduplicated chromosomes and their relation to the structure and molecular composition of this region is briefly discussed.

INTRODUCTION

The regular process of chromosome replication and segregation of sister chromatids keeps constant the karyotype number and structure of any species. This process can be spontaneously or experimentally altered resulting in cells with different ploidy composition.

One of these mechanisms of mitotic modification is endoreduplication. When this mitotic modification occurs, the chromosome set replicates two or more times between mitosis. If the centromere fails to divide, a diplochromosome is formed holding together four chromatids. In case of two consecutive replications, chromosomes appear consisting of two sister chromosomes lying side by side, showing a different picture from the one usually found in tetraploid metaphases where homologues are mixed up in the metaphase plate.

Endomitosis, which is another related mechanism of increasing chromosome number, and endoreduplication are thought to be closely related processes which give rise to somatic polyploidy referred to as endocycles by Nagl (1978). Endoreduplication is a widespread process in plant and animal tissues (D'Amato, 1952; Geitler, 1953). Endoreduplication and endomitosis are usually observed in normal cells, organic tissues and cancer cells (Levan and Hauschka, 1953; Levan and Hsu, 1961; Schwarzacher and Schnedl, 1965; Oksala and Therman, 1974; Sarto et al., 1982). Endoreduplication is also related to the production of giant nuclei in neoplastic cells and seems to be the main process of polyploidization in cancer cells (see Therman et al., 1983a, 1986 and Therman and Kuhn, 1989).

Endoreduplicated chromosomes are also observed in the Chinese hamster established ovary (CHO) cell line which indicates that some mitotic instability is still operating in the in vitro environment. They can also be the result of cell suffering due to adverse culture conditions. In our material we found about 8% endoreduplicated metaphases.

Although it is possible to induce endoreduplication by X-rays (Bell and Baker, 1965) and chemicals, such as adenine (Edwards et al., 1995), Colcemid (Herreros et al., 1966), aphidicolin (Huang et al., 1983), mercaptopyruvate (Jackson and Lindahl-Kiessling, 1963), 33258 Hoechst and rubidazone (Kusyk and Hsu, 1979), colchicine (Rizzoni and Palitti, 1973), hydrazine (Speit et al., 1984), and several DNA intercalating agents, antimetabolites and antitumor antibiotics (Nasjleti and Spencer, 1966, 1967; Sutou and Tokuyama, 1974; Sutou and Arai, 1975), the mitotic mechanism that originates endoreduplication is still obscure (Therman et al., 1983b).

Matsumoto and Ohta (1994) reported the interesting observation that a combination of rotenone and ammonium vanadate treatment induces quadruple and octuple chromosome configurations in CHO-K1 cells, and Takanari et al. (1991) described partial endoreduplication and segmentary endoreduplicated chromosomes together with other chromosome changes produced by vincristine.

In a previous study, we reported that the high density chromatin of the T-banded subtelomeric chromosomes of normal human and CHO chromosomes showed specific distribution patterns, namely: a) similar size in both sister chromatids; b) predominating in one of the chromatids, and c) concentrated in only one chromatid. Pixel count corresponding to the highest sorted absorbance values allowed the following quantitative classification: a) when the interchromatid difference of highest absorbance values was less than 25%, the distribution was considered as identical and identified as Type 1; b) differences of 25 to 75% were classified as Type 2, and c) differences higher than 75% as Type 3. Besides, minute sister chromatid exchanges of densely stained areas were also detected by microphotometrical scanning (Drets et al., 1992a).

Endoreduplicated chromosomes offer a unique opportunity to confirm these patterns and to observe if they are able to replicate reproducing the same chromosome structure in sister chromosomes in terms of high density stain distribution as found in normal human and CHO chromosomes.

In the present study, we confirmed the presence of the three different high density patterns (Types 1-3) in the subtelomeric region of T-banded endoreduplicated CHO chromosomes through microphotometric scanning showing that they replicate similarly in the sister endoreduplicated chromosomes. Also, the highest density chromatin areas were considered as chromosome marker segments, allowing the detection of reciprocal changes of position in endoreduplicated chromosomes when spreading on the slide, a cytogenetic fact not yet reported.

MATERIAL AND METHODS

Cell cultures

CHO cells were grown as monolayers at 37^°C in flasks or Petri dishes in Mc Coy's 5A medium supplemented with 10% fetal calf serum, 200 mM glutamine and antibiotics (penicillin 100 units/ml and streptomycin 0.1 mg/ml) in an atmosphere of 5% CO₂ in air. Cells were harvested after 2 h exposure to Colcemid (0.08 µg/ml; Ciba) using routine methods.

T-banding

Dutrillaux's method (1973) for T-banding was used with the following modifications: 1) slides with air dried CHO metaphases were immediately frozen and stored at -20^°C; 2) slides were defrosted and transferred to an incubator at 60^°C for 4 h before T-banding treatment; 3) CHO cells were incubated in hot (87 ± 0.1^°C) 0.1 M sodium phosphate-citric acid buffer, pH 5.1, for 8-10 min; 4) slides were washed in buffer solution at room temperature for 1 min and in bidistilled water for 5 min; 5) metaphases were stained with Giemsa prepared by dissolving 1.5 ml of Giemsa stock solution (Merck) in 50 ml bidistilled water containing 1.5 ml methanol and 1.5 ml sodium phosphate buffer at pH 6.8.

Scanning microscope photometer instrumentation and computer procedures

A Zeiss microscope photometer MSP65 with a 0.25 µm-step fast scanning stage and Zeiss Luminar lens (25 mm 1:3.5/A 0.15) was used for scanning photomicrographs of T-banded chromosomes. The electronic Zeiss microprocessor was associated "on line" to a DEC MicroVAX 3300 computer (Digital Equipment Corp.) and a TEK Netstation 4211 (Tektronix Inc.) which was used as a graphic color computer terminal. Color photographs were taken directly from the terminal screen. Luminous field and photometric field diaphragm diameters were 0.3 mm and 0.1 mm, respectively. A Zeiss LD Planachromatic objective (40/0.60 - 160/1.1-1.5) was used as microscope condenser.

Scanning of T-banded regions in endoreduplicated CHO chromosomes

T-banded endoreduplicated chromosomes were photographed using a Zeiss Photomicroscope II with a Neofluar oil immersion lens (100X) and phase contrast. Technical Pan film (Kodak) was exposed at a rating of DIN 12 and developed with Microdol (Kodak) at 20°C for 9 min. Negatives were enlarged with a Durst Professional enlarger (Model DA900) and Componon (Schneider) lens with NA 1:3.5 on Fine Grain Positive Film (Kodak) and developed with Dektol (Kodak) for 2 min at 20^°C. Film enlargements thus obtained were used for T-banded segment scanning and computer analysis. Only CHO chromosomes showing clear T-banded terminal segments were scanned, irrespective of chromosome size or centromere position (Figure 1).

Figure 1
- Endoreduplicated CHO metaphase. Several T-banded chromosomes are indicated by arrows. Note the R-banding remnants.

Graphic image processing of T-banded segments

An interactive computer program for image analysis of the subtelomeric region written in a FORTRAN environment was used (Drets et al., 1992a). Each individual endoreduplicated chromosome to be scanned was windowed to obtain appropriate scanning parameters:

Area scanned: 100 meander scanning lines x 100 steps (0.25 µm/step).

Number of measurements per scanning step: 100.

Total number of measurements made by the scanning microphotometer: 1,000,000.

Total number of averaged measurements stored per subtelo-meric chromosome segment: 10,000.

The total number of absorbance measurements (10,000) obtained from each subtelomeric segment scanned was considered sufficient for producing precise computer graphic images. Scanned images corresponded exactly, from a cytogenetical point of view, to the chromatin density stain distribution in this chromosome segment. For local feature detection, T-banded chromosome density values were sorted into 12 classes ranging from one to 100 absorbance units (see color bar and absorbance values in Figure 2c). Pixeled images were displayed on a 0-100 scale corresponding to the absorbance measurement limits of the Zeiss system. Picture element size corresponded approximately to the light microscope resolving power (1 pixel = ~ 0.2 µm). All chromosome scannings were carried out in the same technical conditions since the instrumental settings were included in the computer program to function by default. The system proved to be fast and reliable taking only a few minutes to obtain informative graphic images. The time consumed during scanning and computer procedures was:

Subtelomeric scanning and data acquisition: 3.5 min.

Data transfer to the TEK terminal: 15 s.

Two-D image transformation: 5 s.

Image zooming 6X: 5 s.

Rubberbanding technique (image interpolation): 70 s.

Rubberbanding technique (calculation of the number of sorted pixels per chromatid): 4 s.

Displayed images were re-processed by default to remove background noise. Binary filtering (shrink and expand operation; James, 1987) reduced unwanted pixels displayed in the background surface originated by cytoplasmic debris, stain precipitates or specks of dust on the photograph. Additionally, this feature allowed the smoothening of the subtelomeric image border by removing pixels forming part of the object boundary.

Since the system generated by default two-D pixeled images of 33 x 33 mm, they were zoomed (6X) to reach a final image screen size of 190 x 190 mm which was used for the illustrations (Figure 2a-d).

Interactive quantitative analysis of sorted color pixels

The number of pixels corresponding to each sorted rank per chromatid was counted by means of a graphic computational procedure which consisted in drawing a graphic line in the displayed image to delimit the chromatids. Lines were drawn using the graphic input rubberbanding feature of the TEK color terminal. Each member of the endoreduplicated T-banded chromosome pair was scanned separately for quantitative analysis. This procedure permitted the counting of the number of pixels corresponding to each sorted high chromatin-stained density detected per subtelomeric T-banded segment. False colors were assigned to pixels to enhance contrast in the graphic image facilitating the detection of the high density areas and thus the quantification of telomeric structural features. Detailed descriptions of the cell procedures as well as the microphotometrical and computer graphics methods used were reported elsewhere (Drets et al., 1992a, 1995b).

Figure 2a-d - Two-D graphic pixeled images of T-banded subtelomeric segments of endoreduplicated CHO chromosomes obtained by microphotometric scanning. a) The areas of high density chromatin detected are of similar size in the sister chromatids of both endoreduplicated chromosomes (density pattern Type 1; see text); b) large density areas appear in the inner chromatids (density pattern Type 2); c) high density regions are seen in the outer and inner chromatids; d) high density regions appear in outer chromatids (c and d correspond to Type 3). Note in a and c that high T-banded densities were also detected by the system in the paracentric areas. The thin yellowish line surrounding the red areas in the graphic images is a photographic effect originating from an interaction between the reactivity of the color film used (Kodak Gold 100, Eastman Kodak) and the light emitted by the phosphorous dots of the terminal screen at the border of both pixeled areas (blue and red) of the zoomed 2-D image. Insets and arrows illustrate the endoreduplicated chromosomes and the T-banded segments scanned, respectively. Color bar and values correspond to sorted absorbances assigned to image pixels. Bar = 1 µm.

RESULTS AND DISCUSSION

Figure 1 shows typical T-banded endoreduplicated CHO chromosomes used for routine scannings. Remnants of R-banding are still observed, since T-bands are reportedly a sub-set of them (Ludeña et al., 1991). Two hundred subtelomeric T-banded segments of chromatids corresponding to 50 endoreduplicated chromosomes were scanned. Two-D graphic images of T-banded endoreduplicated chromosomes showing the distribution of high chromatin densities detected in the subtelomeric segment appear (Figure 2a-d).

The three types of patterns (Types 1-3) of high density distribution described for CHO and normal human chromosomes (Drets et al., 1992a) were also detected in the endoreduplicated chromosomes. High density segments of similar size appeared in the four chromatids (Figure 2a). This configuration (Type 1) was found in 12 endoreduplicated chromosomes.

The highest density chromatin subtelomeric segments appearing as blue pixels (Figure 2a-d) were considered as "marker" chromosome segments, allowing further cytogenetical interpretations of the graphic images. An alternation of high density marker areas in subtelomeric segments of sister chromosomes was also observed (Figure 2c and d). The number of endoreduplicated chromosomes presenting this high density alternation was as follows: a) high density segments only observed in the inner chromatids: 12 endoreduplicated chromosomes (Figure 2b; Type 2); b) only one high density segment in the inner chromatid of one endoreduplicated chromosome and another one in the outer chromatid of the sister chromosome: 16 endoredu-plicated chromosomes (Figure 2c; Type 3), and c) high density segments only observed in the outer chromatids: 10 endoreduplicated chromosomes (Figure 2d; Type 3). This distribution of the marker segments suggests that the replicated sister chromosome can be found positioned differently on the slide.

Figure 3 diagrammatically illustrates a hypothetical interpretation of the different high density marker distributions found in endoreduplicated T-banded chromosomes. The basic assumption is that endoreduplicated chromosomes glided when spread on the slide (Case 1) or suffered rotations originating the characteristic marker alternations observed (Cases 2 and 3). In other words, the homologue chromosome of an endoreduplicated chromosome pair can be seen rotated in a specular manner with respect to its partner. This phenomenon could be detected due to the combination of T-banding and microphotometric scanning procedures used in this study.

Figure 3
- Diagrammatic interpretation of the distribution of the areas of high densitometric found in T-banded endoreduplicated chromosome spreads. Open circle pairs denote the terminal segment of sister chromatids transversally cut. Closed circles inside represent the high density marker area detected by microphotometric scanning. A, Normal chromosome before endoreduplication; B, endoreduplicated chromosomes, and C, chromosome spread. Case 1: Endoreduplicated sister chromosomes presenting a high density marker segment in the outer chromatid and another one in the inner chromatid of sister chromosome, showing that they probably glided when spread (opposite arrows) making the high densities appear in an alternated fashion. Case 2: Clockwise rotation of one endoreduplicated chromosome from B (arrows indicate the rotation direction) showing high density marker areas in the outer chromatids. Case 3: One endoreduplicated chromosome rotated counterclockwise (arrows indicate the rotation direction) presenting the high-density marker areas located in the inner chromatids of endoreduplicated chromosomes. The large open arrow indicates the direction of the cell dropping. ER denotes the endoreduplication process and s indicates the microscope slide plane.

The study of telomeric/subtelomeric areas is becoming of increasing cytological importance since this multifunctional chromosome segment intervenes not only in keeping constant the number and structure of the chromosomes of each species but also in cancer development and cell senescence (for reviews see Blackburn and Greider, 1995; Zakian, 1989).

Chromosome telomeres of mammals and higher plants are composed by repeated DNA sequences. Middle repetitive sequences are also found in subtelomeric segments and the pericentric heterochromatic region of many chromosomes (Blackburn and Szostak, 1984; Meyne et al., 1990). Telomere DNA is divided into structural and functional domains. Immediately adjacent to the telomere repeats are telomere sequences constituting a third structural domain formed by very dynamic and numerous telomere associated sequences (TAS) which are mainly located in the sub-telomeric region (Henderson, 1995). The telomeric region is also a DNA protein complex with proteins that bind specifically to telomeric DNA, capping the chromosome ends thus preventing nucleolytic degradation and end-to-end ligation. Telomere proteins probably affect the accessibility of telomeric DNA to telomerase and interact with other structural or regulatory proteins (for review see Fang and Cech, 1995).

We described a cytogenetical procedure to remove specific portions of the subtelomeric segments of CHO and normal human chromosomes by prolonging the incubation time in T-banding buffer. This specific chromatin removal appeared as tiny holes induced in one or both sister chromatids. In some chromosomes these holes were of similar size while in other cases they were different. Holes were also produced in aberrant CHO chromosomes and in the paracentric region as well (Drets et al., 1992b, 1995a).

The tiny holes were located where the highest density chromatin areas were detected by scanning microphotometry, suggesting that these phenomena are related and not the result of cytological artifacts or computer manipulations. In this connection, the distribution of high density segments found in sister chromatids of endoredu-plicated CHO chromosomes strongly suggests that these patterns are real cytological facts, probably reflecting the underlying chromatin organization (Figure 2).

Biotinilated fluorescent telomeric probes often show signals of variable size, number and position in sister chromatids, resembling the distribution of the high density patterns detected by microphotometric scanning of T-banded chromosomes (Schubert, 1992). Chong et al. (1995) identified and cloned a major protein component of human telomeres (TRF factor), showing by means of immunofluorescent labeling that TRF specifically colocalizes with telomeric DNA at chromosome ends. This observation allowed the authors to demonstrate that the telomeric TTAGGG repeat array forms a specialized nucleoprotein complex. Interestingly, in their photograph illustrating an immunofluorescent labeled metaphase the fluorescent signals appear in both chromatids of similar or different sizes or, in some chromosomes, in only one chromatid.

Although molecular analysis of telomeric/subtelo-meric DNA and associated protein complexes gives very important information about its structure, a clear picture of the eukaryotic metaphase chromosome at a high level of organization is not yet available. It is also not well understood up to now the underlying chromosomal organization corresponding to the banding phenomena (Saitoh and Laemmli, 1994). Allen et al. (1988) described the T-bands of human chromosomes as intricate fibrous structures as seen with electron microscopy, showing that subtelomeric areas of metaphase chromosomes are extremely complex structures. Sumner (1990) pointed out that in the banding phenomena dye accessibility may be modified either by proteins or by the variation of DNA base pair composition (or both) along the genome. The density pattern distribution observed in non-endoreduplicated and endoreduplicated chromosomes may thus contribute to give an insight into the organization of subtelo-meric region that may be dependent on the localized distribution of DNA-protein complexes, possibly reflecting different functional stages of the region. All of this still has to be demonstrated.

ACKNOWLEDGMENTS

We are indebted to G.A. Folle, W. Martinez and E.M. Boccardo for valuable suggestions and critical review of the manuscript. We are also grateful to F.J. Monteverde and P.J. Queirolo for computer program development. Research partially supported by the Program of Development of Basic Sciences (PEDECIBA), Uruguay.

RESUMO

Empregaram-se técnicas de escaneamento microfotométrico e análise de imagem gráfica computadorizada para detectar a distribuição de densidades de cromatina em segmentos subteloméricos de cromossomos endo-reduplicados com bandeamento T de células do ovário do hamster chinês (CHO). Os padrões de densidade de cromatina detectados com este método foram similares aos previamente encontrados em CHO e cromossomos humanos normais. As densidades de cromatina mais elevadas foram consideradas como segmentos marcadores que levaram à detecção de alterações recíprocas de posição de cromossomos endo-reduplicados durante o espalhamento das células na lâmina. O problema dos padrões de densidade de bandeamento T subteloméricos encontrados nos cromossomos endo-reduplicados e sua relação com a estrutura e a composição molecular desta região é discutido brevemente.

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(Received January 5, 1998)

*

Dedicated to Prof. Adayapalam T. Natarajan on the occasion of his 70th birthday.

Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  June 1998

History

• Received
  05 Jan 1998